An enormous amount of interest has been generated in the use of embryonic and adult stem cells for cell replacement therapy and the treatment of disease. The most interest has been generated by embryonic stem cells, whose pleuripotent potential enables them to become any tissue in the body. The mechanisms through which embryonic stem cells differentiate have partially been discovered. However, the appropriate concentration and time of delivery of known growth factors have not been adequately determined. Additionally, it is not known if all appropriate growth factors for a given stem cell have been identified.
The use of adult stem cells has also generated a great deal of interest. Adult stem cells are multipotent, rather than pleuripotent. In other words, they are capable of transforming into a variety of tissue types. They can be used in a similar manner to embryonic stem cells, such as for cell replacement therapy and treatment of disease. Interest in adult stem cells and their differentiation has also increased due to a relatively new theory hypothesizing that cancers contain abnormal adult stem cells that are less susceptible to chemotherapy than the more metabolically active progeny of these cancer stem cells. Therefore, new methods of treating cancer should target the proliferation and differentiation of cancer stem cells, as well as reducing the already differentiated cells.
One major problem in studying either the differentiation of embryonic, adult or cancer stem cells is that the differentiation of daughter cells created following division of the stem cells is not stable. These daughter cells often retain a specific cell phenotype for a few days or a few months, and then fail to show the appropriate chemical composition or morphology. This is especially true with attempts to create neurons from embryonic or adult stem cells. While there are numerous reports of the creation of cells with specific neural markers and neurotransmitter phenotypes, usually with the addition of growth factors or retinoic acid to aid differentiation, often these cells fail to maintain their original neurotransmitter phenotype after a few days in culture or following transplantation into the nervous system.
Embryonal carcinoma cells derived from teratocarcinoma contain pleuripotent stem-like cells capable of differentiating into a variety of cell types, including neural cells. Human embryonal carcinomas may be an alternative cell source of adult stem cells. One of the most promising embryonal carcinomas cell source is the Ntera2/D1 (NT2) cell line. The NT2 cell line is derived from an embryonal teratocarcinoma cell line capable of differentiating into post-mitotic dopaminergic neurons (NT2N) following treatment with retinoic acid (RA). RA differentiated NT2N neurons (i.e., hNT neurons) have been shown to engraft within the central nervous system and have been used successfully in ameliorating the behavioral deficits associated with stroke, spinal cord injury, and traumatic brain injury. Although, these cells are derived from teratocarcinoma cells, they do not form tumors in the striatal environment. However, RA differentiation results in an unstable dopaminergic phenotype, leading to the rapid loss of their dopaminergic phenotype. Additionally, RA-induced differentiation of NT2 cells leads to increased apoptosis of differentiated hNT neurons compared to undifferentiated NT2 cells.
Cell aggregation in suspension culture has a profound effect on growth and differentiation of cells. The use of embryonic stem cell suspension cultures that form embryoid bodies, has proven to be valuable method to study lineage commitment and differentiation of pleuripotent stem cells without the influence exerted by surrounding tissue. Suspension cultures of neural stem cells, which form neurospheres, also have proven to be an important method to study proliferation, multipotent differentiation of neural stem cells, and differentiation of neural progenitors. Similarly, teratocarcinomas form embryoid bodies, and have been used as in vitro models to study differentiation and stem cell development. Cell aggregation can influence differentiation and cell fate determination of embryonal carcinoma P19 cells. It was found that a neuronal phenotype was the most abundant phenotype among aggregated mouse embryonic cells, followed by astrocytes and microglia. Recent studies showed that aggregated NT2 cells form spheres that contain cells with neuronal morphology after RA treatment. Additionally, these spheres generate neurons when they are exposed to growth factors that also stimulate neural stem cells. The distinctive feature of cell aggregation is the three-dimensional arrangement of the cells that creates cell-to-cell interaction resembling a normal cell environment in vivo. It was shown that cell to cell contact can activate signaling pathways such as the protein kinase C (PKC) pathway. However, as mentioned above, these cultures required the use of RA and/or growth factors to achieve these results. Use of RA and growth factors can be undesirable due instability issues with the resulting cells, residual RA and growth factors in the cultures, apoptosis concerns and other issues surrounding the use of these compounds.
What is needed is cell that retains its differentiated phenotype for an extended period of time. It would be highly desirable for the methodology used to produce such cells to avoid the use of RA and/or growth factors for development and differentiation of the cells. It would also be desirable to have a cell line that retains the dopaminergic phenotype. The present invention solves this and other important needs as will be evident in the specification below.